ADP-ribosylation factors (ARFs) are a family of proteins, each about 20 kDa in size and having the ability to bind and hydrolyze GTP. ARFs are also characterized by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin (Kahn et al., J Biol Chem 259:6228-6234, 1984; and Tsai et al., J Biol Chem 263:1768-1772, 198). Some members of the ARF protein family are involved in regulating vesicle transport in cells as diverse as yeast and human cells.
The ARF-like protein (ARL) family is related to ARFs by amino acid sequence homology and, like ARFs, are characterized by their ability to bind and hydrolyze GTP. However, ARLs can be distinguished from ARFs as they do not enhance the ADP-ribosyltransferase activity of cholera toxin.
The invention features an antibody which specifically binds the ARL3 polypeptide having the amino acid sequence of SEQ ID NO:2, which can be encoded by a DNA molecule having the sequence of SEQ ID NO:1. By xe2x80x9cspecifically bindsxe2x80x9d is meant that the antibody binds the ARL3 polypeptide having the sequence of SEQ ID NO:2 but not specifically bind other molecules in that sample. For example, the antibody of the invention will not bind to other members of the yeast ARF and ARL families.
The invention also features a transgenic knockout yeast (e.g., Saccharomyces cerevisiae) having a homozygous disruption in its endogenous ARL3 gene, where the disruption prevents the expression of a functional ARL3 protein and the phenotype of the knockout yeast relative to a yeast having a wild type ARL3 gene includes impaired growth at about 15xc2x0 C. The impaired growth can represent 50, 10, 5, or 1% of the growth of wild type yeast at that temperature. The disruption can include an insertion of a nucleic acid sequence into a wild type ARL3 gene in the genome of a parent yeast. Alternatively, the disruption can include an insertion into a mutated but functional ARL3 gene. In some embodiments, the nucleic acid sequence encodes a polypeptide (e.g., one that confers a selectable phenotype on the transgenic knockout yeast). For example, the parent yeast can be incapable of growth in a medium free of uracil, and the selectable phenotype can be the ability to grow in a medium free of uracil.
The antibody of the invention can be used to isolate and clone genes expressing polypeptides homologous to SEQ ID NO:2. Such an antibody is also useful for quantifying the amount of ARL3 in a sample. The transgenic knockout yeast of the invention is useful for identifying genes which are involved in vesicle transport. Such genes can be identified by their ability to complement the growth defect conferred by disruption of the ARL3 sequence.
Other features or advantages of the present invention will be apparent from the following drawings and detailed description, and also from the claims.
The invention relates to the identification of an expressed yeast ARL3 polypeptide and a nucleic acid which encodes it. The polypeptide and nucleic acid were then used to produce antibodies which specifically bind the ARL3 polypeptide and transgenic knockout yeast with a disruption in the ARL3 gene, respectively.
I. Antibodies
Both polyclonal and monoclonal anti-ARL3 antibodies are within the scope of the invention. Polyclonal anti-ARL3 antibodies can be prepared by immunizing a suitable animal, e.g., a rabbit, with an ARL3 immunogen. The anti-ARL3 antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized ARL3. The antibody molecules directed against ARL3 can be isolated from a mammal (e.g., from the blood of the mammal) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-ARL3 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., Nature 256:495-497, 1975; Kozbor et al. (1983) Immunol Today 4:72, 1983; and Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985. The technology for producing various monoclonal antibody hybridomas is well known (see, e.g., Coligan et al. eds., Current Protocols in Immunology, John Wiley and Sons, Inc., New York, N.Y., 1994). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an ARL3 immunogen, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds ARL3.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-ARL3 monoclonal antibody (see, e.g., Current Protocols in Immunology, supra; Galfre et al. (1977) Nature 266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); and Lerner (1981) Yale J. Biol. Med., 54:387-402. Moreover, the ordinary skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (PEG). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind ARL3, e.g., using a standard ELISA assay.
As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-ARL3 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with ARL3 to thereby isolate immunoglobulin library members that bind ARL3. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPJ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum Antibod Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; and Griffiths et al. EMBO J 12:725-734, 1993.
Additionally, recombinant anti-ARL3 antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, which can be made using standard recombinant DNA techniques, are also within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al., Science 240:1041-1043, 1988; Liu et al., Proc Natl Acad Sci USA 84:3439-3443, 1987; Liu et al., J Immunol 139:3521-3526, 1987; Sun et al., Proc Natl Acad Sci USA 84:214-218, 1987; Nishimura et al., Cancer Res 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985; Shaw et al., J Natl Cancer Inst 80:1553-1559, 1988; Morrison, Science 229:1202-1207, 1988; Oi et al., Bio/Techniques 4:214, 1986; U.S. Pat. No. 5,225,539; Jones et al., Nature 321:552-525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J Immunol 141:4053-4060, 1988.
II. Transgenic Yeasts
The first step in producing the transgenic yeast of this invention is to prepare a DNA sequence (xe2x80x9ctargeting moleculexe2x80x9d) that is capable of specifically disrupting an ARL3 gene in yeast cells carrying that gene and rendering that gene non-functional. The targeting molecule is then used to transfect yeast cells and to disrupt the functional ARL3 genes in those cells.
DNA targeting molecules that are capable, in accordance with this invention, of disrupting a functional ARL3 gene resident in cells may be produced using information and processes well known in the art.
A DNA targeting molecule of the present invention has two functions. Those functions are to integrate at a native resident ARL3 gene (xe2x80x9ctarget gene locusxe2x80x9d) and to disrupt ARL3 gene expression associated with that locus so that no functional ARL3 expression is possible. Those two essential functions depend on two basic structural features of the targeting molecule.
The first basic structural feature of the targeting molecule is a pair of regions that are homologous to chosen regions of the target gene locus. That homology (in terms of both sequence identity and length) causes the targeting molecule to integrate by base pairing mechanisms (xe2x80x9chomologous recombinationxe2x80x9d) at the site chosen in the target gene locus in transfected cells.
Homologous recombination is the rearrangement of DNA segments at a sequence-specific site (or sites) within or between DNA molecules through base-pairing mechanisms. The present invention relates to a particular form of homologous recombination sometimes called xe2x80x9cgene targetingxe2x80x9d. In gene targeting, an exogenous xe2x80x9ctargeting moleculexe2x80x9d (or xe2x80x9ctargeting fragmentxe2x80x9d) is introduced into cells. The targeting molecule has one or more regions of homology with a chromosomal gene to be modified or replaced (xe2x80x9ctarget genexe2x80x9d). The regions of homology between the target gene and the targeting molecule result in site-specific integration of the exogenous sequence. Of course, the exogenous sequence may be designed to correct an existing defect in the resident gene or to disable (xe2x80x9cdisruptxe2x80x9d) a functional resident gene. The present invention relates to disrupting ARL3 genes. Gene targeting, which affects the structure of a specific gene already in a cell, is to be distinguished from other forms of stable transformation wherein integration of foreign DNA for expression is not site-specific, and thus does not predictably affect the structure of any particular gene already in the cell.
The second basic structural feature of the targeting molecule of this invention is a disrupting sequence between the homologous regions. The disrupting sequence prevents expression of functional protein from the ARL3 target gene following the replacement of portion of that target gene by the integrated targeting molecule.
One of skill in the art will recognize that numerous embodiments of the ARL3 gene targeting molecule of the present invention may be constructed to fulfill the structural and functional requirements specified above. The example below describes the actual construction of an ARL3 gene targeting molecule used to produce the transgenic yeast of the present invention. The following discussion sets forth considerations and parameters that can be used to design other ARL3 gene targeting molecules.
Parameters of the targeting molecule that may be varied in the practice of the present invention include the lengths of the homologous regions, what regions of the target gene locus are to be duplicated as the homologous regions of the targeting molecule, the length of the disrupting sequence, the identity of the disrupting sequence, and what sequence of the target gene is to be replaced by the targeting molecule.
The length of the homologous regions that flank the disrupting sequence of the targeting molecules can vary considerably without significant effect on practice of the invention. The homologous flanking regions must be of sufficient length for effective heteroduplex formation between one strand of the targeting molecule and one strand of a transfected cell""s chromosome, at the ARL3 target gene locus. Increasing the length of the homologous regions promotes heteroduplex formation and thus targeting efficiency. However, it will be appreciated that the incremental targeting efficiency accruing per additional homologous base pair eventually diminishes and is offset by practical difficulties in targeting molecule construction, as homologous regions exceed several thousand base pairs. An effect range for the length of each homologous region is 50 to 5,000 base pairs, with about 500 base pairs being desirable. It should be further noted that the precise length of the homologous regions in the DNA targeting molecule may depend in practice on the location of restriction sites in and around the ARL3 gene. For a discussion of the length of homology required for gene targeting, see Hasty et al., Mol Cell Biol 11:5586-91, 1991.
There is considerable latitude in choice of which regions of the target gene locus are duplicated as the homologous regions in the targeting molecule. The basic constraints are that the ARL3 target gene sequence to be replaced by the disrupting region must lie between the regions of the target gene locus duplicated as the homologous regions in the targeting molecule, and that replacement of the target gene sequence must render the ARL3 gene non-functional. It should be noted that the target gene locus nucleotide sequences chosen for homology in the targeting molecule remain unchanged after integration of the targeting molecule. Those sequences of the target gene locus are merely replaced by the duplicate (homologous) sequences in the targeting molecule. Identity between the chosen regions of the target gene locus and the homologous regions in the targeting molecule is the means by which the targeting molecule delivers the disrupting sequence precisely into the ARL3 target gene. The chosen regions of homology may lie within the ARL3 coding sequence, but it is not necessary that they do so. For example, in an embodiment of the present invention, one homologous region could be located 5xe2x80x2 from the ARL3 gene, and the other homologous region could be located 3xe2x80x2 from the ARL3 gene. The ARL3 initiation codon and 5xe2x80x2 terminal region of the ARL3 coding sequence can lie between the chosen homologous regions and thus be replaced by the interrupting sequence, so that no portion of the protein can be expressed. When the interrupting sequence contains a selectable marker (or any other gene), there can be a termination codon downstream of the minimum required marker coding sequence, and in-frame with the marker coding sequence, to prevent translational read-through that might yield an ARL3 fusion protein with ARL3 activity. As a practical matter, other than the requirement that some critical site of the ARL3 gene lie between the homologous regions (so that it will be disrupted), the primary constraints on choice of homologous regions is the availability of the cloned sequences and the existence of restriction sites therein. Preferably, the regions chosen to be homologous regions will not include sequences longer than about 20 nucleotides that are known to occur elsewhere in the genome being modified. Extensive homology between the targeting molecule and other (non-target) sites in the genome might diminish targeting efficiency by diverting targeting molecules into non-productive heteroduplexes at non-target sites.
The length of the disrupting sequence separating the homologous regions in the targeting molecule can also vary considerably without significant effect on the practice of the present invention. The minimum length of the disrupting sequence is one base pair. Insertion of a single base pair in the ARL3 coding sequence would constitute a frame shift mutation and thus could prevent expression of a functional protein. Alternatively, a single base pair substitution could result in an amino acid substitution at a critical site in the protein and the expression of only non-functional protein. It should be recognized, however, that a single base pair alteration is susceptible to reversion to the wild type sequence through spontaneous mutation. For that reason, disrupting sequences longer than one base pair are sometimes more useful. At the other extreme, excessive length in the disrupting sequence would be unlikely to confer any advantage over a disrupting sequence of moderate length, and might diminish efficiency of transfection or targeting. Excessive length in this context is many times longer than the distance between the chosen homologous regions on the target gene. The length for the disrupting sequence can be from 2 to 2,000 base pairs. Alternatively, the length for the disrupting sequence is a length approximately equivalent to the distance between the regions of the target gene locus that match the homologous regions in the targeting molecule.
There is wide latitude in the choice of the disrupting sequence, since the disrupting function is not sequence-specific. It is necessary, however, that the nucleotide sequence of the disrupting region not express a functional ARL and not express a protein or polypeptide toxic to the transformed cell. The disrupting sequence should also not be extensively homologous to sites in the genome of the transfected cell. Such homology would be likely to diminish the efficiency of the targeting molecule, and might severely impair its function.
For some embodiments of the present invention it is preferred that the disrupting sequence have a dual function, i.e., be both a selectable marker and a disrupting sequence. In those embodiments, the length and identity of the disrupting sequence will be determined largely by the selectable marker coding sequence and associated expression control sequences. The selectable marker gene provides for positive selection of transfected cells that have taken up and integrated the targeting molecule. The need for a selectable marker will depend on the methods chosen for transfection of cells and transgenic yeast production. The choice of those methods, in turn, will depend on the species of yeast on which this invention is being practiced. Selectable markers include the antibiotic resistance gene, neomycin phosphotransferase (xe2x80x9cneoxe2x80x9d), or thymidine kinase, dihydrofolate reductase, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, adenosine deaminase, asparagine synthetase and CAD (carbamyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase).
In this discussion, the targeting molecule is described as a linear DNA molecule. However, it should be recognized that a targeting molecule of the present invention could also be embodied as a circular DNA molecule. A circular targeting molecule can include a pair of homologous regions separated by a disrupting region, as described for a linear targeting molecule. Alternatively, a circular targeting molecule can include a single homologous region. Upon integration at the target gene locus, the circular molecule would become linearized, with a portion of the homologous region at each end. Thus, the single homologous region effectively becomes two homologous regions, as described in Watson et al., Molecular Biology of the Gene (4th Ed.), Benjamin/Cummings, Menlo Park, Calif., p. 606. One differing aspect of a circular targeting molecule with a single homologous region is that it inserts the disrupting sequence into the target gene and disrupts it without replacing any of the target gene. A second differing aspect is that the single homologous region must be within the target gene and located 5xe2x80x2 to at least one critical site in the ARL3 coding sequence.
A transgenic yeast having a homozygous disruption in its ARL3 gene and exhibiting a observable phenotype due to the ARL3 disruption can be used to identify other genes which function in biochemical pathways affected by ARL3 protein. For example, an ARL3 knockout transgenic yeast exhibits a growth defect at 15xc2x0 C. A library of DNA vectors which encode yeast polypeptides are then introduced into these knockout yeast. A transfected knockout yeast which exhibits wild-type growth at 15xc2x0 C. can be inferred to contain a vector encoding a protein which complements the temperature-sensitive growth phenotype. Therefore, an analysis of the polypeptide encoded by that vector allows the identification of a gene or protein which is involved in vesicle transport.
Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure and the isolation of ARL polypeptides and nucleic acids described below, utilize the present invention to its fullest extent. The following example is to be construed as merely illustrative of how one skilled in the art can isolate ARL polypeptides or nucleic acids from biological sources, and are not limitative of the remainder of the disclosure in any way. Any publications cited in this disclosure are hereby incorporated by reference.